JP5349838B2 - Small RNA acquisition carrier, acquisition method and acquisition reagent - Google Patents

Abstract

An object of the present invention is to provide a carrier which enables to obtain a small RNA in high yield yet in high purity, a simple method for obtaining a small RNA and/or a target nucleic acid of small RNA using the same, and a reagent and a kit comprising the above-described carrier. The present invention relates to (1) a carrier for obtaining a small RNA produced by introducing a polymerizable functional group or a chain transfer group to the surface of a core particle, mixing said particle with a polymerizable monomer, then promoting polymerization reaction to form a layer comprising a polymer compound on the surface of said particle, and immobilizing a physiologically active substance having affinity to a small RNA-binding protein on the surface of the particle; (2) a method for obtaining a small RNA, characterized by using the above-described carrier; (3) a reagent for obtaining a small RNA and/or a target nucleic acid of small RNA, comprising the above-described carrier; (4) a reagent kit for obtaining a small RNA and/or a target nucleic acid of small RNA, comprising said reagent; and (5) a method for obtaining a target nucleic acid of small RNA, characterized by using the above-described carrier.

Description

  The present invention relates to a carrier for obtaining small RNA and a method for obtaining a small RNA and / or a small RNA target nucleic acid using the same.

Micro RNA (miRNA) is a group of functional small RNA consisting of about 22 bases, and has attracted attention as a guide molecule that controls gene expression after transcription. In particular, it is now known to be deeply involved in cell differentiation and canceration. Furthermore, miRNAs form a protein complex called RISC (RNA-induced silencing complex) after being matured through multiple steps in the cell. It has been reported that miRNAs incorporated into protein complexes seek out target mRNAs depending on their base sequences and suppress cleavage or translation.
On the other hand, Ago2 protein, which is a major component of RISC, is known to bind to miRNA, and anti-Ago2 antibody is also commercially available as an antibody that recognizes Ago2 protein. A method for obtaining miRNA by immunoprecipitation using the anti-Ago2 antibody is reported in Ikeda et.al. Journal of lmmunological Methods, 10417, 2006 and the like. However, the anti-Ago2 antibody immobilization carrier used here binds the anti-Ago2 antibody using affinity with Protein A, and the anti-Ago2 antibody elutes depending on the reaction conditions. In addition to the Protein A-immobilized carrier, there is a problem that a complicated operation is required in which anti-Ago2 antibodies must be prepared and reacted with RISC. Therefore, there has been a demand for a stable anti-Ago2 antibody-immobilized carrier that does not require such a complicated operation and does not elute the anti-Ago2 antibody. In addition, since the immunoprecipitation method using the anti-Ago2 antibody-immobilized carrier has just started, development of a method for obtaining small RNA such as miRNA with high yield and high purity using the method. Was also desired.
Ikeda et.al. Journal of lmmunological Methods, 10417, 2006

  The present invention relates to a carrier capable of obtaining small RNA with high yield and high purity, and a simple target RNA for small RNA and / or small RNA (hereinafter abbreviated as small RNA target nucleic acid). It is an object of the present invention to provide a reagent and a kit comprising the above carrier.

  In view of the above situation, the present invention is an invention made by intensive studies by the present inventors. (1) A polymerizable functional group or a chain transfer group is introduced on the surface of a particle serving as a nucleus, and the particle is polymerizable. By mixing the monomer and then proceeding with the polymerization reaction, the particle surface is formed into a particle having a layer containing a polymer compound, and a physiologically active substance having affinity for small RNA binding protein on the surface. A carrier for obtaining small RNA, wherein (2) a method for obtaining small RNA using the carrier, (3) for obtaining small RNA and / or a small RNA target nucleic acid comprising the carrier A reagent, (4) a reagent kit for obtaining small RNA or / and a small RNA target nucleic acid comprising the reagent, and (5) a method for obtaining a small RNA target nucleic acid using the carrier.

  In the carrier of the present invention, for example, the coherent proteins in the cell lysate are hardly adsorbed on the surface thereof, so that the non-specific binding of these proteins causes the binding reaction between the physiologically active substance on the carrier surface and the small RNA binding protein. Inhibition (for example, inhibition of antigen-antibody reaction between Ago2 and anti-Ago2 antibody) hardly occurs. In addition, since there is little non-specific adsorption of protein, it is easy to confirm the recovery of small RNA binding protein (for example, Ago2) from the surface of the carrier. Furthermore, since the inhibition factor due to these non-specifically adsorbed proteins does not increase during RNA purification, according to the method for obtaining small RNA of the present invention using the carrier of the present invention, high yield of small RNA can be obtained. And can be obtained with high purity. Furthermore, according to the method for obtaining small RNA of the present invention, it is possible to omit the step of immobilizing a physiologically active substance such as an antibody on a carrier as compared with the conventional method, and it is used to prevent nonspecific adsorption. There is no need to add a blocking agent or the like, and small RNA can be obtained by a simple operation. Furthermore, if the carrier of the present invention is used, a small RNA target nucleic acid can be easily and efficiently acquired by the same operation as the method for acquiring small RNA.

  The particles according to the present invention are prepared by introducing a polymerizable functional group or chain transfer group to the surface of the core particle, mixing the particle and the polymerizable monomer, and then proceeding the polymerization reaction, thereby causing the core particle surface Are particles in which a layer containing a polymer compound is formed. The polymerizable monomer contains an ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing a physiologically active substance, and further contains an ethylenically unsaturated polymerizable monomer (b) if necessary. It is preferable. The polymer compound formed on the surface of the particle serving as the nucleus has a property of immobilizing a specific physiologically active substance. Moreover, the ethylenically unsaturated polymerizable monomer (b) having an alkyleneoxy group has a property of suppressing nonspecific adsorption of proteins and the like. Furthermore, in order to form a polymer compound on the particle surface using a polymerizable functional group or chain transfer group that forms a covalent bond with the core particle surface, the polymer compound is densely formed on the core particle surface. It can be grafted. The grafted particles obtained in this way will not be washed out by the washing step.

  Examples of the functional group of the ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing a physiologically active substance according to the present invention include a chemically active group, a receptor group, and a ligand group. It is not limited to these. Specific examples include aldehyde groups, active ester groups, epoxy groups, vinyl sulfone groups, biotin derived groups, thiol groups, amino groups, isocyanate groups, isothiocyanate groups, hydroxyl groups, acrylate groups, maleimide groups, hydrazide groups. , Azide group, amide group, sulfonate group, group derived from streptavidin, metal chelate and the like. Among these, an aldehyde group, an active ester group, an epoxy group, and a vinyl sulfone group are preferable from the viewpoint of reactivity with amino groups contained in a large amount of physiologically active substances, and biotin-derived groups are preferable because of high binding constants with physiologically active substances. Is preferred. Of these, an active ester group is most preferable from the viewpoint of storage stability of the monomer.

  Although the ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing a physiologically active substance according to the present invention is not particularly limited, a (meth) acryl group represented by the following general formula [1] And an active ester group are preferably a compound in which an alkyleneoxy group having 1 to 10 carbon atoms is bonded via a chain or an alkylene group. In particular, the alkyleneoxy group linkage itself has the property of suppressing nonspecific adsorption of proteins. For this reason, a monomer in which a (meth) acryl group and an active ester group are bonded via a chain of alkyleneoxy groups has both the property of immobilizing a physiologically active substance and the property of suppressing nonspecific adsorption of proteins. Therefore, even if the polymer of such a monomer is a single polymer, it is suitable as a polymer compound that forms a layer on the particle surface as long as it has a reactive functional group at least on one end. Can be used.

(Wherein R ₁ represents a hydrogen atom or a methyl group, X represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents an active ester group, and p represents an integer of 1 to 100. p) When X is an integer of 2 or more and 100 or less, the repeated Xs may be the same or different.
In the formula [1], the alkyleneoxy group and the alkylene group X have 1 to 10 carbon atoms, preferably 1 to 6, more preferably 2 to 4, still more preferably 2 to 3, most preferably Preferably it is 2. The repeating number p of the alkyleneoxy group and the alkylene group X is an integer of 1 to 100, more preferably an integer of 2 to 90, and most preferably an integer of 2 to 80. In addition, when p is 2 or more and 100 or less, the carbon number of p alkyleneoxy groups or alkylene groups that are repeated may be the same or different.

  The alkylene group may be linear, branched or cyclic, and specifically includes, for example, a methylene group, an ethylene group, a propylene group, a trimethylene group, a butylene group, a 1-ethylethylene group, and 2-methyl. A trimethylene group, a 2-ethyltrimethylene group, a hexylene group, a cyclopropylene group, a cyclobutylene group, a cyclopentylene group, a cyclohexylene group, and the like can be given. Among them, an ethylene group is preferable. Examples of the alkyleneoxy group include those in which an oxy group is bonded to the above alkylene group, and among them, an ethyleneoxy group is preferable. Of the above, the alkyleneoxy group and the alkylene group X of the formula [1] are preferably an alkyleneoxy group, and particularly preferably an ethyleneoxy group.

  The “active ester group” used in the present invention is an ester group having a highly acidic electron-withdrawing group in one of the substituents of the ester group and activated for nucleophilic reaction, ie, the reaction activity. As meaning a high ester group, it is commonly used in the fields of various chemical syntheses such as polymer chemistry and peptide synthesis. Actually, for example, it is an ester group produced by dehydration condensation between a carboxyl group in a certain compound and a compound having a hydroxyl group or a mercapto group. Esters produced by introduction of ester groups include phenolic esters, thiophenol esters, N-hydroxyamine esters, heterocyclic hydroxy compound esters, etc., which are much more active than alkyl esters. Known as having a group.

  Examples of such active ester groups include p-nitrophenyl active ester groups, N-hydroxysuccinimide active ester groups, phthalic acid imide active ester groups, 5-norbornene-2, 3-dicarboximide active ester groups, and the like. Among them, a p-nitrophenyl active ester group or an N-hydroxysuccinimide active ester group is preferable, and a p-nitrophenyl active ester group is most preferable.

  The composition ratio in the polymer compound of the monomer unit derived from the ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing the physiologically active substance according to the present invention is not particularly limited, 1-99.7 mol% is preferable, More preferably, it is 1-80 mol%, Most preferably, it is 1-70 mol%.

  The ethylenically unsaturated polymerizable monomer (b) having an alkyleneoxy group used in the present invention is not particularly limited in structure, but is represented by the general formula [2], a (meth) acryl group and a carbon number of 1 to 10 It is preferable that it is a compound which consists of a chain of alkyleneoxy group Y.

(Wherein R ₂ represents a hydrogen atom or a methyl group, R ₃ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, Y represents an alkyleneoxy group having 1 to 10 carbon atoms, and q represents 1 to 100) (The repeated Y may be the same or different.)
Carbon number of alkyleneoxy group Y in Formula [2] is 1-10, Preferably it is 1-6, More preferably, it is 2-4, More preferably, it is 2-3, Most preferably, it is 2. It is. The repeating number q of the alkyleneoxy group Y is not particularly limited, but is preferably an integer of 1 to 100, more preferably an integer of 2 to 100, and still more preferably an integer of 2 to 95. Most preferably, it is an integer of 20 to 90. When the number of repetitions is 2 or more and 100 or less, the carbon number of q alkyleneoxy groups Y to be repeated may be the same or different.

  Examples of the ethylenically unsaturated polymerizable monomer (b) having an alkyleneoxy group include methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) acrylate, and 2-hydroxypropyl (meth) ) Acrylate and monosubstituted ester of hydroxyl group thereof, 2-hydroxybutyl (meth) acrylate and monosubstituted ester of hydroxyl group thereof, glycerol mono (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, 2-methoxyethyl ( (Meth) acrylate, 2-ethoxyethyl (meth) acrylate, methoxydiethylene glycol (meth) acrylate, ethoxydiethylene glycol (meth) acrylate, ethoxypolyethylene Glycol (meth) acrylate and the like can be mentioned, but methoxypolyethylene glycol methacrylate or ethoxypolyethyleneglycol methacrylate is preferable, and methoxypolyethyleneglycol methacrylate is more preferable because of less nonspecific adsorption of components other than the target physiologically active substance and availability. preferable.

  The composition ratio of the monomer unit derived from the ethylenically unsaturated polymerizable monomer (b) having an alkyleneoxy group in the present invention is not particularly limited, but is preferably 0.3 to 99 mol%. More preferably, it is 20 to 99 mol%, and most preferably 30 to 99 mol%.

  Examples of the polymerizable functional group to be introduced on the particle surface according to the present invention include a vinyl group, an allyl group, a methacryl group, an epoxy group, and a styrene group, and a methacryl group is preferable in terms of excellent polymerizability.

  Examples of the chain transfer group to be introduced on the particle surface according to the present invention include a mercapto group and an amino group, and a mercapto group is preferable in terms of excellent reactivity.

  The method for introducing a polymerizable functional group or chain transfer group onto the particle surface is not particularly limited, but a covalent bond between a silane coupling agent having a polymerizable functional group or chain transfer group and a functional group on the particle surface serving as a nucleus. The method of forming is preferable.

  Examples of the silane coupling agent having a polymerizable functional group include (3-methacryloxypropyl) dimethylmethoxysilane, (3-methacryloxypropyl) diethylmethoxysilane, (3-methacryloxypropyl) dimethylethoxysilane, (3 -Methacryloxypropyl) diethylethoxysilane, (3-methacryloxypropyl) methyldimethoxysilane, (3-methacryloxypropyl) ethyldimethoxysilane, (3-methacryloxypropyl) methyldiethoxysilane, (3-methacryloxypropyl) Ethyldiethoxysilane, (3-methacryloxypropyl) trimethoxysilane, (3-methacryloxypropyl) triethoxysilane, and the like can be mentioned. From the viewpoint of reactivity and availability, (3-methacryloxypropyl) tri Tokishishiran or (3-methacryloxypropyl) triethoxysilane are preferred. These silane coupling agents are used alone or in combination of two or more.

  Examples of the silane coupling agent having a chain transfer group include (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl) methyldimethoxysilane, (3-mercaptopropyl) dimethylmethoxysilane, and (3-mercaptopropyl) tri. Ethoxysilane, (3-mercaptopropyl) methyldiethoxysilane, (3-mercaptopropyl) dimethylethoxysilane, (mercaptomethyl) trimethoxysilane, (mercaptomethyl) methyldimethoxysilane, (mercaptomethyl) dimethylmethoxysilane, (mercapto) Methyl) triethoxysilane, (mercaptomethyl) methyldiethoxysilane, (mercaptomethyl) dimethylethoxysilane, and the like. From the availability, (3-mercaptopropyl) trimethoxysilane and (3-Mercaptopropyl) triethoxysilane is preferred. These mercaptosilane compounds are used alone or in combination of two or more. These silane coupling agents are used alone or in combination of two or more.

  The method for forming a covalent bond between a polymerizable functional group or chain transfer group and a functional group on the particle surface as a core using a silane coupling agent having a polymerizable functional group or chain transfer group is not particularly limited. However, for example, a silane coupling agent having a polymerizable functional group or chain transfer group is added to an acidic aqueous solution having a pH of 2 to 4 so as to be 0.01 to 1.0 mol / L, and the mixture is stirred and mixed to hydrolyze. The particles are added and stirred at 10 to 100 ° C. for 5 to 180 minutes, and then heated to 20 to 100 ° C. to dry the particles. The use ratio of the core particle and the polymerizable functional group or chain transfer group-containing silane coupling agent is not particularly limited, but the polymerizable functional group or chain transfer group is usually added to 1 g of the core particle. The silane coupling agent is used at a ratio of 0.1 to 10 mmol. Although acidic aqueous solution is not specifically limited, Acetic acid aqueous solution, hydrochloric acid aqueous solution, etc. are used. Among these, an acetic acid aqueous solution that is relatively easy to handle is preferable.

  The method of introducing a polymerizable functional group or chain transfer group on the surface of the core particle, mixing the particle and the polymerizable monomer, and then proceeding the polymerization reaction is not particularly limited. For example, the polymerizable monomer In addition, the core particles are put into a solvent in which the polymerization initiator is dissolved and heated at 0 to 80 ° C. for 1 to 30 hours with stirring. Thereafter, the particles whose surface is coated with the polymer compound are filtered under reduced pressure, washed and dried.

  The use ratio of the core particles, the polymerizable monomer, and the polymerization initiator is not particularly limited, but the ratio of the polymerizable monomer 0.1 to 10 mmol and the polymerization initiator 0.01 to 10 mmol with respect to 1 g of the normal core particle. Used in

  Any solvent may be used as long as each polymerizable monomer can be dissolved. For example, methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol and other alcohols, benzene, toluene, tetrahydrofuran, dioxane, List dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, etc. Can do. These solvents are used alone or in combination of two or more.

  The polymerization initiator is not particularly limited, and examples thereof include azo compounds such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and peroxides. Examples thereof include organic peroxides such as benzoyl and lauryl peroxide.

  The chemical structure of the polymer compound used on the particle surface according to the present invention is a (co) polymer containing at least a monomer unit derived from an ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a physiologically active substance. If so, the bonding method may be random, block, graft, or the like.

  The material of the core particle used in the present invention is not particularly limited and can be used regardless of whether it is organic or inorganic, but has (or can be introduced) a group capable of reacting with a silane coupling agent. Those are preferred. As organic carrier, in addition to porous agarose particles (trade name: Sepharose) and dextran particles (trade name: Sephadex), which are used as carriers for affinity chromatography, polyacrylamide gel (trade name: Bio-Gel P Biorad), polystyrene, ethylene-maleic anhydride copolymer, polymethyl methacrylate, and the like. On the other hand, as the inorganic substance, an inorganic oxide is preferable because the particle itself has high strength. Among these, silicon oxide is most preferable because it is easy to handle. The size of the particles is not limited at all, and can be appropriately selected according to the purpose and use. This means that particles of any size can be produced by selecting the size of the core particle. This is a significant advantage compared to methods in which particles are prepared by emulsion polymerization or suspension polymerization, which makes it difficult to control the particle size. When actually used as particles, the particle size is preferably from several nm to about 100 μm, although it varies depending on the application.

  The particles according to the present invention are obtained as described above, and are obtained by adding a component containing an alkyleneoxy group (alkylene glycol residue) to a component of a layer containing a polymer compound on the particle surface. The property of suppressing nonspecific adsorption by components other than proteins is enhanced. Moreover, in order to form a polymer compound using a polymerizable functional group or chain transfer group that forms a covalent bond with the core particle surface, the polymer compound is grafted onto the core particle surface at a high density. Is possible. The grafted particles thus obtained are excellent particles that have extremely low non-specific adsorption and that the polymer compound does not flow out by the washing step.

  The small RNA binding protein according to the present invention varies depending on the type of RNA to be targeted, and examples thereof include a ribosome protein, a snRNP protein, a snRNP protein, and an Argonatute family protein. Among them, the Argonaute family protein is preferable. Specific examples of Argonaute family proteins include the Argonaute subfamily such as Ago1, Ago2, Ago3, and Ago4, and the Piwi subfamily such as PIWIL1, PIWIL2, PIWIL3, PIWIL4, MILI, and MIWI. Argoute subfamily such as Ago2, Ago3, Ago4, etc. is preferable, and Ago2 that binds to siRNA and microRNA (miRNA) is particularly preferable.

  Examples of the small RNA to which the small RNA binding protein according to the present invention binds include 5SrRNA, tRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), siRNA, piRNA, miRNA, etc. Of these, piRNA, siRNA, miRNA and the like are preferable, siRNA, miRNA and the like are more preferable, and miRNA and the like are particularly preferable. The length is usually 5 to 200 nt, preferably 10 to 50 nt, more preferably 10 to 30 nt.

  A physiologically active substance having an affinity for a small RNA binding protein (hereinafter sometimes abbreviated as a physiologically active substance according to the present invention) differs depending on the type of the small RNA binding protein, Examples thereof include antibodies and aptamers having affinity (binding ability), and antibodies are preferred. Specifically, for example, when Ago2 is used as a small RNA binding protein, an anti-Ago2 antibody is used, and when Ago3 is used, an anti-Ago3 antibody is used.

The origin of the antibody used as the physiologically active substance according to the present invention is not particularly limited, and it may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. These antibodies may be commercially available, or may be used as F (ab ′) ₂ , Fab ′, or Fab after digestion with an enzyme such as pepsin or papain if necessary.

As a method for obtaining the above polyclonal antibody, the measurement object is determined according to the method described in [Introduction to Immunological Experiments, Second Printing, Nao Matsuhashi, Society Publishing Center, 1981], for example, horse, cow, sheep, rabbit. It may be prepared by a conventional method for immunizing animals such as goats, guinea pigs, rats and mice. In addition, as a method for obtaining a monoclonal antibody, immunized cells such as spleen cells and lymphocytes of animals such as rats and mice immunized with the measurement object as an immunogen, and myeloma cells and the like A hybridoma is prepared by fusing the cells having the property of permanently proliferating with a cell fusion technique (Nature, 256, 495, 1975) known per se developed by Keller and Milstein et al. A hybridoma that produces a specific monoclonal antibody is selected, and the hybridoma is cultured in a medium or administered into the abdominal cavity of an animal to produce antibodies in ascites, and the desired monoclonal antibody is produced from the culture or ascites The antibody having the above-mentioned properties is produced by a method known per se (Eur. J. Immunol., 6, 511, 1976). Cells were prepared for, and a method of harvesting the desired monoclonal antibodies are exemplified by culturing the cells.
As the aptamer production method, the method described in US Pat. No. 5,270,163 is preferred.

  The amount of the physiologically active substance according to the present invention immobilized on the carrier for obtaining small RNA of the present invention (hereinafter sometimes abbreviated as the carrier of the present invention) varies depending on the type of physiologically active substance used. The amount is usually 0.1 to 10 mg, preferably 1 to 10 mg per 1 g.

The carrier of the present invention is obtained by immobilizing the physiologically active substance according to the present invention on the surface of the particle according to the present invention, and is a carrier used in the method for obtaining small RNA of the present invention. The method for immobilizing the physiologically active substance according to the present invention on the surface of the particle according to the present invention can be carried out by a known immobilization method, for example, a method of immobilizing by a chemical bond such as a covalent bond, or by physical adsorption. Immobilization methods such as Japanese Patent Publication No. 5-41946 may be used, but a method of immobilization by chemical bonding is preferred, and in particular, a physiologically active substance introduced on the particle surface is immobilized. A method of immobilizing using a functional group is particularly preferred. Specifically, for example, 0.5 mL of a solution containing the physiologically active substance according to the present invention usually 2 μg / mL to 200 μg / mL, preferably 20 μg / mL to 200 μg / mL, and 10 mg of the particles according to the present invention are appropriate if necessary. In the presence of a condensing agent, usually at 20 to 50 ° C., preferably at 30 to 40 ° C. for 1 to 20 hours, preferably 1 to 10 hours, more preferably 2 to 5 hours. The carrier of the present invention in which the active substance is immobilized on the surface can be obtained. Here, if necessary, the condensing agent used may be appropriately selected from those used in conventional methods in this field. Further, after immobilizing the physiologically active substance, treatment with a blocking agent such as ethanolamine may be performed to inactivate the functional group on the particle surface according to the present invention where the physiologically active substance is not immobilized. desirable.
The solvent for preparing the solution of the physiologically active substance according to the present invention may be any solvent as long as it does not prevent the physiologically active substance used from adsorbing or binding to the insoluble carrier. For example, purified water such as pH Preferable examples include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, sodium bicarbonate buffer and the like having a buffering action at 5.0 to 10.0, preferably pH 8.5 to 10. The buffer concentration in these buffers is appropriately selected from the range of usually 0.1-5 M, preferably 0.6-2.5 M. In addition, this solution contains, for example, saccharides, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as they do not prevent the antibody from adsorbing or binding to the insoluble carrier. Also good.
In addition, since the particle | grains based on this invention have few nonspecific adsorption | suctions, it is not necessary to attach | subject the support | carrier of this invention obtained as mentioned above to the blocking process normally performed in this field | area.

  Specifically, the carrier of the present invention is produced as follows.

  That is, for example, 1 to 10 g of silica beads are added to an acetic acid aqueous solution (for example, pH 2 to 4) of 0.01 to 1.0 mol / L of a silane coupling agent, and reacted at 50 to 100 ° C. for 10 to 180 minutes to obtain a polymerizable functional group. Silica beads with groups or chain transfer groups are made. On the other hand, the monomer represented by the general formula [1] and the monomer represented by the general formula [2] are mixed so as to have a ratio of, for example, 1:99 to 70:30, and dissolved in a solvent such as dehydrated ethanol. Next, 1 to 10 times mol of a polymerization initiator such as AIBN is added to the solution and stirred to obtain a monomer mixed solution. Silica beads are added to the monomer mixed solution so that the polymer is present in an amount of 0.1 to 10 mmol with respect to 1 g of the silica beads having the polymerizable functional group or chain transfer group, 50 to 80 ° C., 10 to 30 hours, If necessary, the particles according to the present invention can be obtained by reacting in an argon atmosphere and drying. Subsequently, 0.1 g to 10 mg of the physiologically active substance according to the present invention such as anti-Ago2 antibody is present per 1 g of the particles, and the sodium bicarbonate buffer containing the physiologically active substance according to the present invention is added to the particles according to the present invention. By adding it and reacting at 30 to 40 ° C. for 2 to 10 hours, the physiologically active substance according to the present invention is immobilized on the surface of the particles, and the carrier of the present invention is obtained.

  The carrier of the present invention has little non-specific adsorption, and is a complex of small RNA binding protein and small RNA. Furthermore, when small RNA binds to a target nucleic acid (for example, when small RNA is miRNA or piRNA) ), Can specifically adsorb complexes of small RNA binding protein, small RNA and small RNA target nucleic acid (hereinafter, these may be abbreviated as a small RNA binding protein complex) . If the carrier non-specifically adsorbs a protein other than the small RNA binding protein complex, it inhibits the binding of the small RNA binding protein complex etc. to the carrier due to the adsorbed protein. Steric hindrance). Therefore, the recovery rate of small RNA binding protein complex etc. falls. However, since the carrier of the present invention is unlikely to cause such steric hindrance and the like, according to the method using the carrier, a small RNA binding protein complex or the like can be recovered in a high yield, and as a result For example, small RNA can be obtained in high yield.

The method for obtaining small RNA of the present invention is a method for obtaining small RNA by using the carrier of the present invention obtained as described above, and specifically comprises the following steps.
That is, step (1): introducing a polymerizable functional group or a chain transfer group to the surface of the core particle, mixing the particle and the polymerizable monomer, and then proceeding the polymerization reaction, thereby increasing the surface of the particle. A carrier in which a physiologically active substance having affinity for a small RNA binding protein is immobilized on the surface of a particle on which a layer containing a molecular compound is formed, and a complex of the small RNA binding protein and small RNA (small RNA binding) A protein-small RNA complex) and a conjugate of the physiologically active substance and the small RNA binding protein-small RNA complex (hereinafter referred to as physiologically active substance-small RNA binding protein-small RNA conjugate) on the surface of the carrier. Step (2): separating the obtained carrier having the physiologically active substance-small RNA binding protein-small RNA conjugate on the surface, step (3): Said physiologically active substance Eluting the small RNA from small RNA-binding protein -small RNA bound substance, and the step (4): comprising the step of purifying the eluted small RNA.

Specifically, first, the carrier of the present invention is added to a solution containing a small RNA binding protein-small RNA complex, and the temperature is 2 to 37 ° C., preferably 2 to 10 ° C. for 1 to 30 hours, preferably 2 to 24. By reacting for a time, a complex of the physiologically active substance and the small RNA binding protein-small RNA complex is formed on the surface of the carrier of the present invention (step (1)). The small RNA binding protein-small RNA complex includes a complex in which the small RNA binding protein according to the present invention and the small RNA according to the present invention are bound, and a protein that binds to the complex (for example, Gemin3 , Gemin4, FMRP, etc.). Specific examples of the complex include, for example, RISC (RNA-induced silencing complex). Examples of the solution containing a small RNA binding protein-small RNA complex used in this case include a cell lysate (cell extract) containing a small RNA binding protein-small RNA complex, and the lysate is usually a solution. One containing 5 × 10 ⁶ to 1 × 10 ⁷ cells in 1 mL is used. Examples of the solvent (reaction solvent) for the solution containing the small RNA binding protein-small RNA complex include all cell lysates usually used in this field. Specifically, for example, a buffer solution containing a surfactant and NaCl, etc. Is mentioned. Examples of the surfactant include poly (oxyethylene) nonylphenyl ether (NP-40), and the concentration thereof is usually 0.01 to 0.5% with respect to the total amount of the buffer solution. The concentration of NaCl is usually 100 to 200 mM as the concentration in the buffer. Further, as the buffer solution, for example, pH 5.0 to 10.0, preferably having a buffering action near neutral pH 6.5 to 8.5, for example, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution. A liquid or the like is preferable. The buffer concentration in the buffer is appropriately selected from the range of usually 10 to 500 mM, preferably 10 to 300 mM. The buffer containing the surfactant and NaCl may be added with TritonX-100, SDS, or the like in a concentration usually used in this field, if necessary. The amount of the carrier of the present invention used in the step (1) is usually 1 to 10 mg, preferably 1 to 5 mg per 1 mL of the small RNA binding protein-small RNA complex solution. The amount of the active substance is usually 0.1 to 100 μg, preferably 1 to 50 μg.

  Next, after the reaction in the above step (1), the carrier having the physiologically active substance-small RNA binding protein-small RNA binding substance on the surface is separated from the obtained reaction solution (step (2)). Specifically, the reaction solution of (1) is centrifuged and then the supernatant is removed, whereby the carrier of the present invention is separated from the reaction solution of (1). Furthermore, in order to remove free small RNA binding protein-small RNA complex and cell-derived components adhering to the surface of the carrier, it is preferable to wash the obtained carrier with a washing solution. The centrifugation is not particularly limited as long as it is usually carried out in this field, but the operation of centrifuging the carrier of the present invention suspended in an appropriate washing solution at, for example, 1000 to 10000 × g for 10 to 100 seconds. Preferably, it may be performed several times. Examples of the washing solution include buffers generally used in this field. For example, pH 5.0 to 10.0, preferably pH 6.5 to 8.5 has a buffering action near neutrality, such as phosphate buffer, Tris buffer, Good Examples thereof include a buffer solution, a glycine buffer solution, and a borate buffer solution. The buffer concentration in the buffer is appropriately selected from the range of usually 10 to 500 mM, preferably 10 to 300 mM. In addition, it is preferable to use the same cleaning liquid as the reaction solvent in step (1). The amount of the cleaning liquid used is usually 1 to 10 times the amount of the reaction liquid in the above step (1).

  Subsequently, after separating the carrier having the bound substance on the surface in the step (2), the small RNA is eluted from the physiologically active substance-small RNA binding protein-small RNA bound substance on the surface of the carrier. Specifically, a protein denaturing agent is added to a carrier having the bound substance on the surface to denature the physiologically active substance. Thereby, the physiologically active substance and the small RNA binding protein are denatured, and the small RNA is separated and eluted (step (3)). The protein denaturing agent used here may be any that can denature the physiologically active substance according to the present invention. For example, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, urea, guanidine hydrochloride, guanidine thiocyanate, citric acid. Examples thereof include a buffer solution (pH 2 to pH 4). Among them, SDS is preferable. The amount used is 10 to 1000 μL, preferably 10 to 400 μL, per 1 to 5 mg of carrier.

  Furthermore, the small RNA is separated by purifying the small RNA eluted in the step (3) by a nucleic acid extraction method or the like (step (4)). Any nucleic acid extraction method can be used as long as it is usually used in this field, and examples thereof include extraction with a mixed solution of phenol / chloroform / isoamyl alcohol. The ratio of the mixed solution is usually phenol: chloroform: isoamyl alcohol = 25: 24: 1, and the amount used is the same as the elution solution. The small RNA obtained as described above is more preferably purified by a method such as alcohol precipitation, column purification, filter filtration or the like usually used in this field.

More specifically, the method for obtaining small RNA of the present invention is as follows. That is, for example, 1 to 10 mg, preferably 1 to 5 mg of the carrier of the present invention is added to 1 mL of 5 × 10 ⁶ to 1 × 10 ⁷ cell lysate containing a complex of Ago2 and small RNA, and 2 to 2 at 10 ° C. React for 24 hours. Furthermore, after centrifuging the obtained reaction solution at 3000 to 5000 × g for 10 to 50 seconds and removing the supernatant, 1 to 5 mL of Tris buffer (pH 6.5 to 8.5) with respect to 1 mL of the reaction solution, The carrier to which the complex is bound is washed several times. Next, 10 to 400 μL of SDS solution is added to 1 to 5 mg of the carrier of the present invention, and small RNA is eluted from the carrier. Furthermore, 1 μL of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to 1 μL of the elution solution, and small RNA was extracted. Then, 2 μL to 3 μL of ethanol and 0.05 μL of sodium acetate were added to 1 μL of the extraction solution. Purified small RNA is obtained as a precipitate by adding 0.005-0.01 μL of a coprecipitation agent such as 0.1 μL, etaprecipitate, glycogen or the like.

  In the method for obtaining small RNA of the present invention, a carrier in which a physiologically active substance is immobilized on the particle surface (the carrier of the present invention) is used, so that a physiologically active substance such as an antibody is immobilized on the carrier as compared with the conventional method. It is possible to omit the step of conversion. Further, there is no need to add a blocking agent used to prevent nonspecific adsorption, purification is easy, and small RNA and the like can be obtained by a simple operation.

Some small RNAs bind to nucleic acids as targets. By utilizing the property, the target nucleic acid of small RNA can be obtained by using the carrier of the present invention. The target nucleic acid may be DNA or RNA as long as it is targeted by small RNA, and the chain length is not particularly limited. The target here means that small RNA has affinity, and specifically, it is preferable to bind complementarily. In the case of complementary binding, not all strands need to be complementary, but at least 6 bases or more of the small RNA and the target nucleic acid must be complementary. As the target nucleic acid, for example, when small RNA is microRNA, RNA is preferable, and mRNA is more preferable. In addition, when small RNA is piRNA, RNA is preferable, but when DNA can be a target nucleic acid, it may be DNA.
The method for obtaining the target nucleic acid of small RNA is as follows.
That is, since the small RNA having the above properties is bound to the target nucleic acid in the cell, preferably, by performing the steps (1), (2) and (3) of the method for obtaining small RNA of the present invention, By performing steps (1), (2), (3) and (4), a conjugate of small RNA and its target nucleic acid can be obtained. Therefore, the obtained small RNA and its target nucleic acid conjugate are separated by adding a solution such as formamide, and subjected to electrophoresis such as a denaturing acrylamide gel to have the target nucleotide (nt). By extracting the nucleic acid, the nucleic acid targeted by the small RNA can be obtained. The small RNA target nucleic acid obtained in this way has a normal concentration. Therefore, when the small RNA target nucleic acid is DNA, the obtained nucleic acid may be subjected to PCR reaction to be amplified. Since the concentration of the target nucleic acid in the conjugate of the obtained small RNA and the target nucleic acid obtained above is low, the conjugate may be directly subjected to PCR reaction to be amplified. The PCR reaction conditions are usually set according to the conditions used in this field.
When the target nucleic acid is RNA, the target nucleic acid cDNA is obtained by subjecting a conjugate of the small RNA obtained as described above and the target nucleic acid to a reverse transcription reaction using a primer corresponding to the target nucleic acid. It may be amplified by subjecting it to a PCR reaction as in the case of the above DNA. The conditions for the reverse transcription reaction are usually set according to the conditions used in this field. In addition, by sequencing the DNA obtained as described above, it becomes possible to specify the target nucleic acid of small RNA. Furthermore, it is also possible to analyze effects such as cleavage or translational inhibition on the target nucleic acid by a small RNA binding protein-small RNA complex or the like.

  The present invention will be described in more detail with reference to the following examples and reference examples, but the present invention is not limited to these examples.

Experimental Example 1 Production of Mouse IgG Immobilization Carrier Using Particles According to the Present Invention (1) Production of Protein Immobilization Particles (Beads) [Synthesis of p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP)]
0.01 mol of polyethylene glycol monomethacrylate (Blenmer PE-200 manufactured by NOF Corporation) was dissolved in 20 mL of chloroform, and then cooled to -30 ° C. To this solution while maintaining at -30 ° C, 0.01 mol of p-nitrophenylchloroformate (Aldrich), 0.01 mol of triethylamine (Wako Pure Chemical Industries, Ltd.) and 20 mL of chloroform were prepared. The homogeneous solution of was slowly added dropwise. After reacting at −30 ° C. for 1 hour, the solution was further stirred at room temperature for 2 hours. Thereafter, the salt was removed from the reaction solution by filtration, and the solvent was distilled off to obtain a crude p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (hereinafter referred to as MEONP). Furthermore, the obtained crude product was purified with a silica gel column. The obtained monomer was measured by 1H-NMR in deuterated chloroform solvent, and it was confirmed that 4.5 units of ethylene glycol residue was contained.

[Preparation of silica beads treated with methacryloxypropyl trimethoxysilane]
7.45 g of methacryloxypropyltrimethoxysilane (LS3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to 39.3 g of an acetic acid aqueous solution having a pH of 3.0 and stirred at room temperature for 1 hour. Thereto, 5 g of silica beads (average particle size 5 μm, pore size 70 mm, SMB70-5 manufactured by Fuji Silysia Chemical Co., Ltd.) was added and stirred at 85 ° C. for 2 hours, and then the silica beads were recovered from the reaction solution by suction filtration. Heated at 100 ° C. for 1 hour. Then, disperse with ethanol, shake for 1 hour at room temperature, repeat the operation of removing the supernatant by centrifugation twice, and further disperse with ethanol, stir with a vortex mixer, and remove the supernatant by centrifugation Was repeated 5 times and dried.

[Preparation of protein immobilization particles (beads)]
Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate, hereinafter referred to as PEGMA475, manufactured by Aldrich) having a number average molecular weight Mn of about 475 and MEONP obtained above were dissolved in dehydrated ethanol to prepare a monomer mixed solution. The total monomer concentration was 0.2 mol / L, and each molar ratio was PEGMA475: MEONP = 80: 20. Azobisisobutyronitrile (AIBN) was added thereto so as to be 0.004 mol / L, and the mixture was stirred until uniform. Thereafter, 1 g of silica beads treated with the above methacryloxypropyl trimethoxysilane was added and reacted at 60 ° C. for 22 hours under an argon gas atmosphere. Then, the silica beads were recovered from the reaction solution by suction filtration, and ethanol was added. The operation of dispersing and removing the supernatant by centrifugation was repeated 5 times, and then the beads were collected by suction filtration and dried well.

(2) Preparation of mouse IgG immobilized carrier
[Preparation of mouse IgG solution]
1 mg of mouse IgG (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 1 mL of phosphate buffer (PBS, manufactured by Wako Pure Chemical Industries, Ltd., pH 7.4) to give a 1 mg / mL mouse IgG solution.

[Preparation of mouse IgG immobilized carrier]
10 mg of the protein immobilization particles obtained in (1) above, 50 μL of mouse IgG solution, and 500 μL of protein immobilization buffer (Sumitomo Bakelite Co., Ltd.) were mixed and mixed by inverting at 37 ° C. for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette and washed twice with 500 μL of PBS (pH 7.4). Thereafter, 500 μL of an inactivation buffer solution (manufactured by Sumitomo Bakelite Co., Ltd.) was added and mixed by inverting at room temperature for 1 hour. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette, washed 5 times with 500 μL of PBS (pH 7.4), and suspended in 1 mL of PBS (pH 7.4).

Comparative Example 1 Preparation of mouse IgG antibody-immobilized carrier using various carriers (1) Immobilization of mouse IgG on GE Healthcare carrier NHS-activated Sepharose 4 Fast Flow (GE Healthcare) was used. According to the recommended protocol, mouse IgG antibody was immobilized as follows. That is, NHS-activated Sepharose 4 Fast Flow 100 μL was washed twice with 500 μL of ice-cold 1 mM hydrochloric acid, 50 μL of mouse IgG solution, and 500 μL of coupling buffer (0.5 M sodium chloride pH 8.3 containing 0.2 M sodium bicarbonate). Were added and mixed, and mixed by inverting at room temperature for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant is removed with a pipette, washed with 500 μL of coupling buffer, and then quenched with buffer (0.5 M ethanolamine, 0.5 M sodium chloride (pH 8.3)) ) 500 μL was added and mixed by inversion for 30 minutes at room temperature. After centrifugation (3000 × g, 1 minute), the supernatant is removed with a pipette, washed three times alternately with 500 μL of quenching buffer and 500 μL of washing solution (0.5 M sodium chloride pH 4 containing 0.1 M acetic acid), and then PBS ( Washing was performed 3 times with 500 μL of pH 7.4) and suspended in 1 mL of PBS (pH 7.4).

(2) Immobilization of mouse IgG on Pierce carrier Mouse IgG (Wako Pure Chemical Industries, Ltd.) was used as a carrier according to the recommended protocol using an immunoprecipitation kit (Seize-Primary Mammalian Immunoprecipitation Kit, Pierce). ) Was immobilized. After washing 100 μL of the kit component beads twice with 400 μL of coupling buffer (Pierce), add 50 μL of mouse IgG solution, 200 μL of coupling buffer, 4 μL of 5 M sodium cyanoborohydride (Pierce) and mix. The mixture was mixed by inverting at room temperature for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant is removed with a pipette and washed with 400 μL of coupling buffer, followed by 400 μL of quenching buffer (Pierce) and 4 μL of 5M sodium cyanoborohydride. The mixture was mixed by inverting at room temperature for 30 minutes. After centrifugation (3000 × g, 1 minute), the supernatant is removed with a pipette, washed 6 times with 400 μL of washing solution (Pierce), then washed 3 times with 400 μL of PBS (pH 7.4), and PBS (pH 7. 4) Suspended in 1 mL.

(3) Immobilization of mouse IgG on Dynal carrier Dynabeads M-270 Carboxylic Acid (DYNAL) was used as a carrier and mouse IgG antibody was immobilized as follows according to the recommended protocol. After washing 100 μL of Dynabeads M-270 Carboxylic Acid with 500 μL of reaction buffer (0.1 M MES pH 5.0) three times, 50 μL of mouse IgG solution and 500 μL of reaction buffer were added and mixed, and mixed by inverting at room temperature for 30 minutes. Thereafter, 25 μL of 10 mg / mL water-soluble carbodiimide (WSC) solution was added, and the mixture was mixed by inverting at room temperature overnight. After magnetic separation, the plate was washed 4 times with 500 μL of a washing solution (0.05 w / v% Tween20 (pH 7.4) containing 25 mM Tris-HCl, 150 mM sodium chloride) and suspended in 1 mL of PBS (pH 7.4).

Experimental example 2 Nonspecific adsorption experiment using mouse IgG antibody immobilization carrier using various carriers
[Preparation of cell lysate]
HeLa cells (Dainippon Pharmaceutical Co., Ltd.) in Dullbecco's Modified Eagle's Medium medium (DMEM, manufactured by Wako Pure Chemical Industries, Ltd.) supplemented with fetal bovine serum (FBS, manufactured by Thermo Electron) to a final concentration of 10 v / v% After the culture solution was removed, the plate was washed twice with PBS (-) (PBS containing no calcium or magnesium). Remove the cells from the dish using trypsin / EDTA solution (Wako Pure Chemical Industries, Ltd.), add medium to suppress trypsin activity, centrifuge the culture (1000 × g, 5 minutes), and supernatant Was excluded. The cell pellet was suspended in 1 mL of PBS (−), the number of cells was counted, 1 × 10 ⁷ cells were transferred to a sterilized 1.5 mL tube, centrifuged (1000 × g, 5 minutes), and the supernatant was removed.
Add 1 mL of cell lysate (0.05 w / v% NP-40 (Wako Pure Chemical Industries, Ltd.) containing 20 mM Tris-HCl, 200 mM sodium chloride, 2.5 mM magnesium chloride) to the cell pellet, and pipette the cells. After suspension, the mixture was allowed to stand on ice for 10 minutes. After centrifugation (20000 × g, 20 minutes, 4 ° C.), the supernatant (cell lysate) was collected.

[Non-specific adsorption test of cell-derived protein]
200 μL of the carrier for immobilizing the mouse IgG of the present invention obtained in Experimental Example 1 and the carrier of each company on which the mouse IgG obtained in Comparative Example 1 was immobilized were centrifuged (3000 × g, 30 seconds) or magnetically separated, and then the supernatant was removed. Except for this, 1 mL of cell lysate was added to each, and the mixture was refrigerated overnight by inversion. After centrifugation (3000 × g, 30 seconds) or magnetic separation, the supernatant was removed and washed 3 times with 1 mL of cell lysate. Thereafter, 20 μL of 2 w / v% sodium dodecyl sulfate (SDS) solution was added to the pellet to elute the protein bound to the carrier.
2x SDS sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 10 µL of the eluate, and the sample incubated for 3 minutes at 95 ° C was added to Supersep HG10-20w / v% gel (Wako Pure Chemical Industries, Ltd.) And subjected to SDS polyacrylamide gel electrophoresis (25 mA, 1 hour). Thereafter, the gel was silver-stained using silver dye 2 kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
The results of SDS polyacrylamide gel electrophoresis are shown in FIG.

  As is clear from the results of FIG. 1, the mouse IgG-immobilized carrier using the particles according to the present invention is more non-specifically adsorbable for cell-derived proteins than the carriers obtained by immobilizing mouse IgG obtained in Comparative Example 1. Was found to be low.

Example 1 Preparation of anti-human Ago2 antibody-immobilized carrier
1 mg / mL anti-human Ago2 antibody (manufactured by Wako Pure Chemical Industries, Ltd.) 50 μL, protein immobilization particles 10 mg obtained in Experimental Example 1 (1), protein immobilization buffer (Sumitomo Bakelite Co., Ltd.) 500 μL was mixed and mixed by inverting at 37 ° C. for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette and washed twice with 500 μL of PBS (pH 7.4). Thereafter, 500 μL of an inactivation buffer solution (manufactured by Sumitomo Bakelite Co., Ltd.) was added and mixed by inverting at room temperature for 1 hour. After centrifugation (3000 × g, 1 minute), remove the supernatant with a pipette, wash with 500 μL of PBS (pH 7.4) 5 times, suspend in 1 mL of PBS (pH 7.4), and immobilize anti-human Ago2 antibody A carrier was obtained.

Comparative Example 2 Preparation of anti-human Ago2 antibody-immobilized carrier using various carriers (1) Immobilization of anti-human Ago2 antibody to GE Healthcare carrier 1 mg / mL anti-human Ago2 antibody instead of 50 μL of mouse IgG solution ( Except for using 50 μL of Wako Pure Chemical Industries, Ltd., anti-human Ago2 antibody was used as a carrier in the same manner as the method for immobilizing mouse IgG on a carrier manufactured by GE Healthcare in Comparative Example 1 (1). Immobilized.
(2) Immobilization of mouse IgG and anti-human Ago2 antibody on Pierce carrier Except for using 50 μL of 1 mg / mL anti-human Ago2 antibody (manufactured by Wako Pure Chemical Industries, Ltd.) instead of 50 μL of mouse IgG solution, The anti-human Ago2 antibody was immobilized on the carrier by the same operation as the method for immobilizing mouse IgG on the Pierce carrier of Comparative Example 1 (2).
(3) Immobilization of mouse IgG and anti-human Ago2 antibody on Dynal carrier Comparison except that 50 mg of 1 mg / mL anti-human Ago2 antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of 50 μL of mouse IgG solution The anti-human Ago2 antibody was immobilized on a carrier in the same manner as the method for immobilizing mouse IgG on the Dynal carrier in Example 1 (3).

Experimental Example 3 Specific adsorption experiment using anti-human Ago2 antibody immobilized carrier using various carriers
[Specific adsorption test of cell-derived protein]
200 μL each of the anti-human Ago2 antibody-immobilized carrier solution obtained in Example 1 and the carrier solutions obtained by immobilizing the anti-human Ago2 antibody obtained in Comparative Example 2 were centrifuged (3000 × g, 30 seconds) or after magnetic separation. The supernatant was removed, and 1 mL of the cell lysate obtained in Experimental Example 2 was added to each of the supernatants. After centrifugation (3000 × g, 30 seconds) or magnetic separation, the supernatant was removed and washed 3 times with 1 mL of cell lysate. Thereafter, 20 μL of 2 w / v% sodium dodecyl sulfate (SDS) solution was added to the pellet to elute the protein bound to the carrier.
10 μL of 2 × SDS sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 10 μL of each eluate, and the sample incubated for 3 minutes at 95 ° C. was added to Supersep HG10-20 w / v% gel (Wako Pure Chemical Industries ( Manufactured by Co., Ltd.) and subjected to SDS polyacrylamide gel electrophoresis (25 mA, 1 hour). Thereafter, the gel was silver-stained using silver dye 2 kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
The results of SDS polyacrylamide gel electrophoresis are shown in FIG.

  As is clear from the results of FIG. 2, in the case of the anti-human Ago2 antibody-immobilized carrier using the particles according to the present invention, an Ago2 band of about 100 kDa that was not seen in FIG. I found that it was trapped. In addition, in comparison with the results obtained for various carriers obtained by immobilizing anti-human Ago2 antibody obtained in Comparative Example 2, when the carrier of the present invention was used, the Ago2 band appeared deeply and generally It was also found that the non-specifically adsorbed cell-derived protein band was small and the amount of non-specific adsorbate other than Ago2 was low. That is, it was found that migos bound to Ago2 and Ago2 can be obtained with a high yield by using the carrier.

Example 2 Method for separating miRNA
[Preparation of anti-human Ago2 antibody immobilization solution]
1 mg / mL anti-human Ago2 antibody (manufactured by Wako Pure Chemical Industries, Ltd.) 25, 50, 100 μL, 475, 450, 400 μL of protein immobilization buffer (Sumitomo Bakelite Co., Ltd.) were added and mixed, Solutions for immobilizing 2.5, 5, 10 μg antibody / mg beads were prepared.

[Preparation of anti-human Ago2 antibody immobilization carrier]
10 mg of the protein immobilization particles obtained in Experimental Example 1 (1) were mixed with 500 μL of the various anti-human Ago2 antibody immobilization solutions prepared above, and mixed by inverting at 37 ° C. for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette and washed twice with 500 μL of PBS (pH 7.4). Thereafter, 500 μL of an inactivation buffer solution (manufactured by Sumitomo Bakelite Co., Ltd.) was added and mixed by inverting at room temperature for 1 hour. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette, washed 5 times with 500 μL of PBS (pH 7.4), and suspended in 1 mL of PBS (pH 7.4).

[Preparation of cell lysate]
HeLa cells (Dainippon Pharmaceutical Co., Ltd.) are cultured in DMEM (Wako Pure Chemical Industries, Ltd.) medium supplemented with FBS (Thermo Electron) so that the final concentration is 10v / v%. Then, the plate was washed twice with PBS (−). Remove the cells from the dish using trypsin / EDTA solution (Wako Pure Chemical Industries, Ltd.), add medium to suppress trypsin activity, centrifuge the culture (1000 × g, 5 minutes), and supernatant Was excluded. The cell pellet was suspended in 1 mL of PBS (−), the number of cells was counted, 1 × 10 ⁷ cells were transferred to a sterilized 1.5 mL tube, centrifuged (1000 × g, 5 minutes), and the supernatant was removed.
1 mL of cell lysate (20 mM Tris-HCl, 200 mM sodium chloride, 2.5 mM magnesium chloride, 0.05 w / v% NP-40) was added to the cell pellet, and the cells were suspended by pipetting and allowed to stand on ice for 10 minutes. . After centrifugation (20000 × g, 20 minutes, 4 ° C.), the supernatant (cell lysate) was collected.

[Isolation of cell-derived microRNA (miRNA) by immunoprecipitation]
Centrifugation (3000 xg, 30 seconds) 200 µL of anti-human Ago2 immobilized carrier solution obtained by the above method (3000 × g, 30 seconds), remove supernatant, add 1 mL of cell lysate, and invert overnight Mixed. After centrifugation (3000 × g, 30 seconds), the supernatant was removed and washed 3 times with 1 mL of cell lysate. Thereafter, 40 μL of 2 w / v% SDS solution was added to the pellet to elute the protein bound to the carrier. Sterile water 360 μL and phenol: chloroform: isoamyl alcohol (25: 24: 1) 400 μL were added to the eluate, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, 400 μL of chloroform was added, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, added with Eta precipitated mate (Nippon Gene Co., Ltd.) 3 μL, 3M sodium acetate 40 μL, and ethanol 1 mL, suspended in a vortex mixer, and then centrifuged (20000 × g, 15 minutes). The precipitate was washed with 1 mL of 70 v / v% ethanol, air-dried at room temperature for 20 minutes, dissolved in 10 μL of sterilized water, and used as a sample for electrophoresis.
A denaturing polyacrylamide gel for electrophoresis was prepared as follows. First, 1 mL of 5 × TBE (Nippon Gene Co., Ltd.), 4.8 g of urea, 2.5 mL of 40% polyacrylamide solution (acrylamide: methylenebisacrylamide = 19: 1) and 2.5 mL of sterilized water were mixed, and then TEMED (N, N , N, N-tetramethylethylenediamine) 10 μL and 10% ammonium persulfate 80 μL were added to crosslink the gel.
10 μL of formamide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 5 μL of the sample for electrophoresis, incubated at 80 ° C. for 3 minutes, and then rapidly cooled on ice. The modified polyacrylamide gel (10 w / v% acrylamide / 8 M (Urea / 0.5 × TBE).
As a control, 10 μL of formamide was added to 1 μL each of synthetic RNA oligos (UAGCUUAUCAGACUGAUGUUGA) with a length of 22 nucleotides of 0.25, 0.5, 1, 2 ng / μL, incubated at 80 ° C. for 3 minutes, and then rapidly cooled on ice. Applied to a polyacrylamide gel.
After electrophoresis (10 mA, 1 hour) in 0.5 × TBE buffer, the gel was silver stained using CLEAR STAIN Ag (manufactured by Nippon Gene Co., Ltd.). The result of denaturing polyacrylamide gel electrophoresis is shown in FIG.

  As is clear from the results of FIG. 3, it was found that the miRNA fraction can be isolated with high purity by the immunoprecipitation method using the carrier of the present invention.

Example 3 Method for separating target mRNA corresponding to miRNA
[Preparation of mouse IgG immobilization solution]
1 mg of mouse IgG (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 1 mL of PBS (pH 7.4) to obtain a 1 mg / mL mouse IgG solution. To 50 μL of 1 mg / mL mouse IgG solution, 450 μL of protein immobilization buffer (Sumitomo Bakelite Co., Ltd.) was added and mixed to prepare a 5 μg mouse IgG / mg immobilization solution.

[Preparation of anti-human Ago2 antibody immobilization solution]
To 50 mg of 1 mg / mL anti-human Ago2 antibody (manufactured by Wako Pure Chemical Industries, Ltd.), 450 μL of protein immobilization buffer (Sumitomo Bakelite Co., Ltd.) is added and mixed to fix 5 μg of anti-human Ago2 antibody beads A solution was prepared.

[Preparation of mouse IgG immobilized carrier solution and anti-human Ago2 antibody immobilized carrier solution]
The protein immobilization particles 10 mg obtained in Experimental Example 1 (1) were mixed with the mouse IgG immobilization solution or anti-human Ago2 antibody immobilization solution prepared above, and mixed by inverting at 37 ° C. for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette and washed twice with 500 μL of PBS (pH 7.4). Thereafter, 500 μL of an inactivation buffer solution (manufactured by Sumitomo Bakelite Co., Ltd.) was added and mixed by inverting at room temperature for 1 hour. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette, washed 5 times with 500 μL of PBS (pH 7.4), suspended in 1 mL of PBS (pH 7.4), and mouse IgG was immobilized. A solution containing a carrier was used as a mouse IgG-immobilized carrier solution, and a solution containing a carrier on which an anti-human Ago2 antibody was immobilized was used as an anti-human Ago2 antibody-immobilized carrier solution.

[Purification of RNA derived from HeLa cells by immunoprecipitation]
200 μL each of a mouse IgG-immobilized carrier solution and an anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads) were centrifuged (3000 × g, 30 seconds), and the supernatant was removed. In addition, 1 mL of HeLa cell lysate was added to each, and the mixture was inverted by refrigeration for 3 hours. After centrifugation (3000 × g, 30 seconds), the supernatant was removed and washed 3 times with 1 mL of cell lysate. Thereafter, 50 μL of 0.5 w / v% SDS solution was added to the bead pellet to elute the protein bound to the carrier. 350 μL of sterilized water and 400 μL of phenol: chloroform: isoamyl alcohol (25: 24: 1) were added to the eluate, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, 400 μL of chloroform was added, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, added with Eta precipitated mate (Nippon Gene Co., Ltd.) 3 μL, 3M sodium acetate 40 μL, and ethanol 1 mL, suspended in a vortex mixer, and then centrifuged (20000 × g, 15 minutes). After washing the precipitate with 1 mL of 70 v / v% ethanol, air-dry at room temperature for 20 minutes, dissolve in 10 μL of sterile water to obtain purified RNA, and fix the RNA-containing solution obtained using mouse IgG-immobilized carrier to mouse IgG An RNA solution using an immobilized carrier and an RNA-containing solution obtained using an anti-human Ago2 antibody-immobilized carrier were used as an RNA solution using an anti-human Ago2 antibody-immobilized carrier.

[Purification of total RNA from HeLa cells]
1 mL of ISOGEN (manufactured by Nippon Gene Co., Ltd.) was added to the HeLa cell pellet (1 × 10 ⁷ cells), and the cells were lysed by pipetting, and then 0.2 mL of chloroform was added and mixed with a vortex mixer. The mixture was allowed to stand on ice for 1 hour and then centrifuged (20000 × g, 10 minutes) to separate the upper layer. To the separated solution, 0.6 mL of chloroform was added, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, 0.6 mL of isopropanol was added and mixed, and then centrifuged (20000 × g, 10 minutes). The precipitate was washed with 1 mL of 70 v / v% ethanol, air-dried at room temperature for 20 minutes, and dissolved in 261 μL of sterilized water to obtain a RNA concentration solution of 1 mg / mL.

[Reverse transcription reaction of purified RNA]
Obtained by the above immunoprecipitation method are 10 μL each of an RNA solution using a mouse IgG-immobilized carrier and an RNA solution using an anti-human Ago2 antibody-immobilized carrier, 1 μL of sterile water and 5 mM oligo d (T) _12-18 primer (manufactured by GE Healthcare) ) 1 μL was added and mixed. Further, 10 μL of sterilized water and 1 μL of 5 mM oligo d (T) _12-18 primer (manufactured by GE Healthcare) were added to and mixed with 1 μL of the purified Total RNA solution. After incubating each mixture at 70 ° C for 3 minutes, ice-cooled, 2.5 µm dNTP mixture (Nippon Gene Co., Ltd.) 4 µL, RNase inhibitor super (Wako Pure Chemical Industries, Ltd.) 1 µL, River Script IV 10 × reaction buffer (Wako Pure Chemical Industries, Ltd.) 2 μL, River Script IV (reverse transcriptase, Wako Pure Chemical Industries, Ltd.) 1 μL was added and mixed to prepare a total reaction volume of 20 μL, 42 Incubated for 30 minutes at ° C.

[PCR amplification of human NRAS cDNA]
As primers for PCR amplification of human NRAS cDNA (GenBank Accession: NM_002524), a forward primer (AATAATAGCAAGTCATTTGCGG) and a reverse primer (CCACACATGGCAATCCCATA) synthesized (Sigma Aldrich Japan) were used.
1 μL of each of the above reverse transcription reaction products, 5 μM Forward primer 1 μL, 5 μM Reverse primer 1 μL, 2.5 mM dNTP mixed solution (Takara Bio) 0.8 μL, Ex Taq polymerase 10 × reaction buffer (Takara Bio) 1 μL, Ex Add 0.2 μL of Taq polymerase (Takara Bio) and 5 μL of sterilized water to prepare a total reaction volume of 10 μL, and perform 40 cycles of PCR (95 ° C for 30 seconds, 60 ° C for 30 seconds, 60 ° C for 60 seconds) It was.
Apply 1 μL of loading buffer (Nippon Gene Co., Ltd.) 1 μL to 5 μL of reaction solution, and apply it to 1.5% agarose gel with final concentration of 1 μg / mL ethidium bromide, and add 1 × TAE buffer (Nippon Gene Co., Ltd.) Electrophoresis (100V, 25 minutes) in. Bands were detected on the gel after electrophoresis with UV transilluminator FASIII (manufactured by Toyobo Co., Ltd.).
The results of agarose gel electrophoresis are shown in FIG.

  Johnson et. Al, Cell, Vol. 120, 635-647, 2005, etc. have reported NRAS mRNA as a target mRNA corresponding to let-7 miRNA expressed in HeLa cells. From the results shown in FIG. 4, a cDNA amplification band (302 bp) corresponding to NRAS mRNA was not detected by immunoprecipitation using the particles according to the present invention in which mouse IgG was immobilized, but anti-Ago2 antibody was immobilized. It was detected by the immunoprecipitation method using the particles according to the present invention. That is, it was found that if the anti-Ago2 antibody-immobilized carrier of the present invention is used, a target mRNA of miRNA that binds to Ago2 can also be obtained. Similarly to the result of Experimental Example 2, no amplification band was detected in the immunoprecipitation method using the particles according to the present invention in which mouse IgG was immobilized, and thus there was no nonspecific adsorption of the target mRNA to the carrier. I found out. Therefore, it has been clarified that not only miRNA but also mRNA targeted by miRNA can be obtained by the immunoprecipitation method using the carrier of the present invention on which an anti-Ago2 antibody is immobilized.

Example 4 Analysis of target mRNA corresponding to miRNA
Using HepG2 cells (hepatoma cells) as cells that do not have miR-122 (micro RNA), miR-122 is introduced into the cells, and miR-122 target mRNA (AlodoA and CAT-1) is introduced from the introduced cells. Was obtained using an anti-human AGO2 antibody-immobilized carrier.

[Preparation of miR-122-introduced HepG2 cells and firefly luciferase siRNA (GL3) -introduced HepG2 cells]
HepG2 cells (Dainippon Pharmaceutical Co., Ltd.) in Dullbecco's Modified Eagle's Medium medium (DMEM, Wako Pure Chemical Industries, Ltd.) supplemented with fetal bovine serum (FBS, manufactured by Thermo Electron) to a final concentration of 10v / v% After the culture solution was removed, the plate was washed twice with PBS (-) (PBS containing no calcium or magnesium). Remove the cells from the dish using trypsin / EDTA solution (Wako Pure Chemical Industries, Ltd.), add medium to suppress trypsin activity, centrifuge the culture (1000 × g, 5 minutes), and supernatant Was excluded. After suspending the cell pellet in DMEM medium supplemented with fetal bovine serum at a final concentration of 10 v / v%, the number of cells was counted, and 5 × 10 ⁶ cells were cultured in a 210 cm ² culture flask with 50 mL of DMEM medium supplemented with fetal calf serum at a final concentration of 10 v / v (Made by Corning).
600pmol hsa-miR-122 synthetic double-stranded RNA (5'-UGGAGUGUGACAAUGGUGUUUGU-3 ', 5'-AAACACCAUUGUCACACUCCAUA-3') or 600pmol firefly luciferase siRNA (GL3, manufactured by Nippon Gene) Opti-MEM medium (Invitrogen) ) Mixed with 10 mL. 100 μL of Lipofectamine RNAi max (manufactured by Invitrogen) was added and mixed with each of the obtained two mixed solutions, allowed to stand at room temperature for 20 minutes, added to the culture flask, and cultured at 37 ° C. for 24 hours.
The culture solution was removed from each flask, and then washed twice with PBS (−). Furthermore, after detaching the cells from the dish using trypsin / EDTA solution, add DMEM medium supplemented with fetal bovine serum at a final concentration of 10v / v% to suppress trypsin activity, and centrifuge the culture solution (1000 × g, 5 minutes) The supernatant was removed. Each pellet obtained was suspended in 1 mL of PBS (-), 900 μL was dispensed for immunoprecipitation RNA purification, 100 μL was dispensed for total RNA purification, and centrifuged (1000 × g, 5 minutes) followed by supernatant The miR-122 introduced cell pellet and the GL3 introduced cell pellet for immunoprecipitation RNA purification and total RNA purification were obtained.

[Preparation of anti-human AGO2 antibody immobilization solution]
To 50 mg of 1 mg / mL anti-human AGO2 antibody (Wako Pure Chemical Industries, Ltd.), add 450 μL of protein immobilization buffer (Sumitomo Bakelite Co., Ltd.) and mix to fix 5 μg of anti-human AGO2 antibody beads. A solution was prepared.

[Preparation of anti-human AGO2 antibody-immobilized carrier solution]
The protein immobilization particles 10 mg obtained in Experimental Example 1 (1) and the anti-human AGO2 antibody immobilization solution prepared above were mixed, and mixed by inverting at 37 ° C. for 4 hours. After centrifugation (3000 × g, 1 minute), the supernatant was removed with a pipette and washed twice with 500 μL of PBS (pH 7.4). Thereafter, 500 μL of an inactivation buffer solution (manufactured by Sumitomo Bakelite Co., Ltd.) was added and mixed by inverting at room temperature for 1 hour. After centrifugation (3000 × g, 1 minute), remove the supernatant with a pipette, wash with 500 μL of PBS (pH 7.4) 5 times, suspend in 1 mL of PBS (pH 7.4), and immobilize anti-human AGO2 antibody A carrier solution was obtained.

[Purification of RNA by immunoprecipitation]
The cell lysate (0.05 w / v% NP-40 containing 20 mM Tris-HCl, 200 mM sodium chloride, 2.5 mM magnesium chloride) was added to each miR-122-introduced cell pellet and GL3-introduced cell pellet dispensed for immunoprecipitation RNA purification. 1 mL of Wako Pure Chemical Industries, Ltd.) was added, the cells were suspended by pipetting, and allowed to stand on ice for 10 minutes. Thereafter, the supernatant (cell lysate) was collected after centrifugation (20000 × g, 20 minutes, 4 ° C.).
After centrifugation (3000 xg, 30 seconds) of 200 µL of the above anti-human AGO2 antibody-immobilized carrier solution (3000 xg, 30 seconds), the supernatant was removed, 1 mL of each of the above cell lysates was added, and the mixture was mixed by inverting for 3 hours. . After centrifugation (3000 × g, 30 seconds), the supernatant was removed and washed 3 times with 1 mL of cell lysate. Thereafter, 50 μL of 0.5 w / v% SDS solution was added to the bead pellet to elute the protein bound to the carrier. 350 μL of sterilized water and 400 μL of phenol: chloroform: isoamyl alcohol (25: 24: 1) were added to the eluate, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, 400 μL of chloroform was added, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). The upper layer was separated, added with Eta precipitated mate (Nippon Gene) 3 μL, 3M sodium acetate 40 μL, and ethanol 1 mL, suspended in a vortex mixer, and then centrifuged (20000 × g, 15 minutes). The precipitate is washed with 1 mL of 70 v / v% ethanol, air-dried at room temperature for 20 minutes, dissolved in 50 μL of sterile water, and the immunoprecipitation purified RNA solution derived from miR-122-introduced cells and the immunoprecipitation purified RNA solution derived from GL3 cells Got.

[Purification of total RNA]
Add 1 mL of ISOGEN (Nippon Gene) to each miR-122-introduced cell pellet and GL3 cell pellet dispensed for total RNA purification, lyse cells by pipetting, add 0.2 mL of chloroform, and vortex mixer Mixed with. The mixture was allowed to stand on ice for 1 hour and then centrifuged (20000 × g, 10 minutes) to separate the upper layer. Chloroform 0.6mL was added to each fractionated solution, mixed with a vortex mixer, and then centrifuged (20000 × g, 10 minutes). Thereafter, the upper layer was separated, 0.6 mL of isopropanol was added and mixed, and then centrifuged (20000 × g, 10 minutes). The precipitate was washed with 1 mL of 70 v / v% ethanol, air-dried at room temperature for 20 minutes, and dissolved in 50 μL of sterilized water to obtain a purified RNA solution derived from miR-122-introduced cells and a purified RNA solution derived from GL3 cells.

[Reverse transcription reaction and quantitative PCR of miRNA]
miRNA reverse transcription using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and TaqMan MicroRNA Assay Kit (for hsa-miR-122, hsa-miR-21) (Applied Biosystems) went.
100 mM in 1 μL of 100 μl dilution of 99 μL of purified water added to 1 μL of immunoprecipitation purified RNA derived from miR-122-introduced cells, or 100 μL of 100 μl dilution of 99 μL of purified water in 1 μL of immunoprecipitated purified RNA of GL3 transfected cells dNTP mix 0.15μL, 10 × RT Buffer 0.75μL, 20U / μL RNase Inhibitor 0.19μL, 50U / μL Multiscribe RT enzyme 1μL, 5 × RT primer (for hsa-miR-122 or hsa-miR-21) 3μL, purified water 8.16 μL was added to each to prepare a total of 15 μL of reaction solution, and two reaction solutions were prepared. Each reaction solution was incubated at 16 ° C. for 30 minutes and at 42 ° C. for 30 minutes, and then incubated at 85 ° C. for 5 minutes to obtain a reverse transcription reaction product.
To 1.33 μL of each reverse transcription reaction product, 10 μL of TaqMan 2 × PCR Master Mix (Applied Biosystems), 1 × L of 20 × TaqMan Assay Mix (for hsa-miR-122 or hsa-miR-21), 7.67 μL of purified water After addition, a total 20 μL reaction solution was prepared, and quantitative PCR detection was performed using ABI7500 Fast Real-Time PCR System (manufactured by Applied Biosystems) to determine each Ct value. On the other hand, single-stranded synthetic miR-122 (5'-UGGAGUGUGACAAUGGUGUUUGU-3 ') or single-stranded synthetic miR-21 (5'-UAGCUUAUCAGACUGAUGUUGA-3') is used as standard RNA, and 10 ¹ to 10 ⁵ copies / in purified water. A diluted solution diluted 10-fold in μL was prepared and subjected to reverse transcription reaction in the same manner as described above to perform quantitative PCR detection. Calibration curves of miR-122 and miR-21 were prepared from the Ct value of each diluted standard RNA solution and the number of molecules. Using the calibration curve, the numbers of miR-122 and miR-21 molecules were calculated from the Ct values obtained above. The quantification results for miR-21 and miR-122 derived from miR-122 introduced cells and miR-21 and miR-122 derived from GL3 introduced cells are shown in FIG.
From the results of FIG. 5, it was confirmed that miR-122, which was not found in GL3-introduced cells, was detected in miR-122-introduced cells, and miR-122 was introduced into HepG2 cells.

[Reverse transcription and quantitative PCR of mRNA]
The reverse transcription reaction of mRNA was performed as follows using SuperSript VILO cDNA Synthesis Kit (Invitrogen).
That is, to each 14 μL of the immunoprecipitation purified RNA solution derived from the miR-122-introduced cells or the GL3 cell-derived immunoprecipitation purified RNA solution, 5 × VILO Reaction Mix 4 μL and 10 × SuperScript Mix 2 μL were added and mixed, respectively, for a total of 20 μL. Two kinds of immunoprecipitation purified RNA reaction solutions were prepared. In addition, 2 × L of purified total RNA derived from miR-122-introduced cells and 2 μL of purified total RNA derived from GL3 cells were added and mixed with 5 × VILO Reaction Mix 4 μL, 10 × SuperScript Mix 2 μL, and purified water 12 μL. A total RNA reaction solution was prepared. The immunoprecipitation purified RNA reaction solution and the total RNA reaction solution were incubated at 25 ° C. for 10 minutes and 42 ° C. for 60 minutes, respectively, and then incubated at 85 ° C. for 5 minutes to obtain a reverse transcription reaction product.
On the other hand, a forward primer (GTTGTGGGCATCAAGGT) and a reverse primer (CAATCTTCAGCACACAACG) and a human Cathionic amino acid transporter-1 (CAT-1) cDNA (GenBank Accession: NM_003045) Forward primer (GTTCCAGAGGGAGCATC) and Reverse primer (CAAGCAAATAACTACCAGGTC) as PCR primers for amplification, and Forward primer (AATCCCATCACCATCTTCC) and Reverse primer (GCAGAGATGATGACCCTTT) as PCR primers for amplification of human GAPDH cDNA (GenBank Accession: NM_002046) did.
To each 1 μL of the reverse transcription reaction product of the immunoprecipitation purified RNA reaction solution obtained above, 0.8 μL of 5 μM Forward primer, 0.8 μL of 5 μM Reverse primer for cDNA synthesis, 2 × Power SYBR Master Mix (manufactured by Applied Biosystems) ) Add 10 μL and 7.4 μL of purified water to prepare 20 μL of each reaction solution, perform quantitative PCR detection of each reaction solution using ABI7500 Fast Real-Time PCR System (Applied Biosystems), and miR122 The Ct value of AldoA, the Ct value of CAT-1 and the Ct value of GAPDH derived from the introduced cells, and the Ct value of AldoA, the Ct value of CAT-1 and the Ct value of GAPDH derived from the GL3 introduced cells were obtained. In addition, using each reverse transcription reaction product of the total RNA reaction solution obtained above, quantitative PCR detection was performed in the same manner as the method using the reverse transcription reaction product of the immunoprecipitation purified RNA reaction solution, and miR122 The Ct value of AldoA, the Ct value of CAT-1 and the Ct value of GAPDH derived from the introduced cells, and the Ct value of AldoA, the Ct value of CAT-1 and the Ct value of GAPDH derived from the GL3 introduced cells were obtained.
For each of AldoA, CAT-1 and GAPDH of the immunoprecipitated purified RNA, and for each of AldoA, CAT-1 and GAPDH of the total RNA, the Ct value of the RNA derived from the miR-122-introduced cell from the Ct value of the RNA from the GL3-introduced cell Calculate the value obtained by subtracting the value using the following formula:
log ₂ [-(RNA Ct value derived from GL3 cells-RNA Ct value derived from miR-122 cells)]
The increase / decrease in the amount of each mRNA of the miR-122-introduced cells relative to the GL3-introduced cells was compared. The result is shown in FIG.

According to Joacim et.al., Nucleic Acids Research, 36 (4), 2008, Aldorase A (AldoA) has a miR-122 target seed sequence in the 3′UTR region of mRNA. 122 target mRNAs have been reported. According to Suvendra et.al., Cell, 125, 1111-1124, 2006, cationic amino acid transporter 1 (CAT-1) is also reported as a target mRNA of miR-122. On the other hand, since firefly luciferase siRNA (GL3) is not an miRNA, even if it is introduced into HepG2, it does not bind to mRNAs such as AldoA and CAT-1. Therefore, the GL3-introduced cells in this experiment serve as a control (standard) for miR-122-introduced cells.
As shown in FIG. 6, AldoA and CAT-1 mRNA, which are the targets of miR-122, were miR-122-introduced cells, but the total mRNA amount was decreased compared to GL3-introduced cells. Had increased. On the other hand, GAPDH mRNA without the miR-122 target sequence showed no significant difference in Ct values between total RNA and Ago2 immunoprecipitation RNA. From the above, it can be seen that AldoA and CAT-1 which are target mRNAs of miR-122 can be efficiently obtained by performing the Ago2 immunoprecipitation method of the present invention using HepG2 cells into which miR-122 has been introduced. That is, this result indicates that microRNA and its target mRNA are specifically enriched in Ago2 immunoprecipitated RNA by overexpressing specific microRNA. By using the method of the present invention, the target of microRNA It was revealed that small RNA target nucleic acids such as mRNA can be efficiently obtained, and that target nucleic acids such as mRNA can be analyzed efficiently.

This is a result of gel electrophoresis of a solution obtained by conducting non-specific adsorption experiments by immunoprecipitation using mouse IgG antibody-immobilized carriers using HeLa cells as samples and various carriers. It is the result of gel electrophoresis of the solution obtained by conducting Ago2 adsorption experiments by immunoprecipitation using anti-Ago2 antibody-immobilized carriers prepared from various carriers using HeLa cells as samples. It is the result of carrying out the gel electrophoresis of the solution (miRNA containing solution) obtained by the immunoprecipitation method using the anti-human Ago2 antibody fixed support | carrier of various density | concentrations using HeLa cell as a sample. Included in each solution using total RNA solution of HeLa cells, solution obtained by immunoprecipitation using mouse IgG immobilized carrier, and solution obtained by immunoprecipitation using anti-human Ago2 antibody immobilized carrier It is the result of subjecting RNA to reverse transcription reaction, further subjecting to PCR reaction, and gel electrophoresis of the resulting solution. It is the result of measuring the amount of miR-122 and miR-21 in Ago2 immunoprecipitation RNA obtained from HepG2 cells into which miR-122 double-stranded RNA or firefly luciferase siRNA (GL3) was introduced, by quantitative PCR. miR-122 Ago2 immunoprecipitated RNA obtained from HepG2 cells transfected with double-stranded RNA or firefly luciferase siRNA (GL3), and AldoA, CAT-1, and GAPDH mRNA levels in total RNA were measured by quantitative PCR. .

Claims (16)

A layer containing a polymer compound was formed on the particle surface by introducing a polymerizable functional group or chain transfer group on the surface of the core particle, mixing the particle and a polymerizable monomer, and then proceeding a polymerization reaction. A carrier in which an antibody to a small RNA binding protein selected from Ago1, Ago2, Ago3, Ago4, PIWIL1, PIWIL2, PIWIL3, PIWIL4, MILI and MIWI is chemically immobilized on the surface of the particle ,
The ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing an antibody, wherein the polymerizable monomer is represented by the following general formula [1]

(Wherein R ₁ represents a hydrogen atom or a methyl group, X represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents an active ester group, and p represents an integer of 1 to 100. p) Is an integer of 2 or more and 100 or less, the repeated Xs may be the same or different from each other), and if necessary, further represented by the following general formula [2] An ethylenically unsaturated polymerizable monomer having an alkyleneoxy group (b)

(Wherein R ₂ represents a hydrogen atom or a methyl group, R ₃ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, Y represents an alkyleneoxy group having 1 to 10 carbon atoms, and q represents 1 to 100) The repeated Y may be the same or different.) A carrier for obtaining small RNA. The carrier according to claim 1, wherein the active ester group is a p-nitrophenyl active ester group. The carrier according to claim 1 or 2, wherein the antibody is an antibody against Ago1, Ago2, Ago3, or Ago4 . The carrier according to any one of claims 1 to 3, wherein the ethylenically unsaturated polymerizable monomer (b) having an alkyleneoxy group is methoxypolyethylene glycol (meth) acrylate. The ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing an antibody is p-nitrophenyloxycarbonyl-polyethylene glycol (meth) acrylate, and an ethylenically unsaturated polymerizable monomer having an alkyleneoxy group (b The carrier according to claim 4, wherein) is methoxypolyethylene glycol (meth) acrylate. A method for obtaining small RNA, wherein the carrier according to any one of claims 1 to 5 is used. The acquisition method according to claim 6, comprising the following steps:
(1) Ethylenic unsaturation having a functional group for immobilizing an antibody represented by the following general formula [1] by introducing a polymerizable functional group or chain transfer group into the surface of the core particle Polymerizable monomer (a)

(Wherein R ₁ represents a hydrogen atom or a methyl group, X represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents an active ester group, and p represents an integer of 1 to 100. p) When X is an integer of 2 or more and 100 or less, the repeated Xs may be the same or different, respectively, and if necessary, an alkyleneoxy group represented by the following general formula [2] Ethylene-based unsaturated polymerizable monomer (b)

(Wherein R ₂ represents a hydrogen atom or a methyl group, R ₃ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, Y represents an alkyleneoxy group having 1 to 10 carbon atoms, and q represents 1 to 100) Of the particles in which a layer containing a polymer compound is formed on the surface of the particles by mixing Y, which may be the same or different , and then allowing the polymerization reaction to proceed. Ago1, Ago2, Ago3, Ago4, PIWIL1, PIWIL2, PIWIL3, PIWIL4, a carrier on which an antibody against a small RNA binding protein selected from MILI and MIWI is chemically immobilized, and Ago1, Ago2, Ago3, Ago4, A small RNA binding protein selected from PIWIL1, PIWIL2, PIWIL3, PIWIL4, MILI and MIWI and a small RNA complex (small RNA binding protein-small RNA complex) are contacted, and the antibody and the small RNA are contacted on the surface of the carrier. Binding protein-small RNA complex Form a body bond,
(2) separating the obtained carrier having on its surface a conjugate of the antibody and small RNA binding protein-small RNA complex;
(3) Elute small RNA from the conjugate of the antibody and small RNA binding protein-small RNA complex (4) Purify the eluted small RNA. The acquisition method according to claim 7, wherein the active ester group is a p-nitrophenyl active ester group. The method according to claim 7 or 8, wherein the antibody is an antibody against Ago1, Ago2, Ago3, or Ago4 . The acquisition method according to any one of claims 7 to 9, wherein the ethylenically unsaturated polymerizable monomer (b) having an alkyleneoxy group is methoxypolyethylene glycol (meth) acrylate. The ethylenically unsaturated polymerizable monomer (a) having a functional group for immobilizing an antibody is p-nitrophenyloxycarbonyl-polyethylene glycol (meth) acrylate, and an ethylenically unsaturated polymerizable monomer having an alkyleneoxy group (b The acquisition method of Claim 10 whose) is methoxypolyethyleneglycol (meth) acrylate. A reagent for obtaining a target nucleic acid for small RNA and / or small RNA, comprising the carrier according to claim 7. A reagent kit for obtaining small RNA and / or a target nucleic acid for small RNA, comprising the reagent according to claim 12. A method for obtaining a target nucleic acid of small RNA, comprising using the carrier according to any one of claims 1 to 5. The acquisition method according to claim 14, comprising the following steps;
(1) Ethylenic unsaturation having a functional group for immobilizing an antibody represented by the following general formula [1] by introducing a polymerizable functional group or chain transfer group into the surface of the core particle Polymerizable monomer (a)

(Wherein R ₁ represents a hydrogen atom or a methyl group, X represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents an active ester group, and p represents an integer of 1 to 100. p) When X is an integer of 2 or more and 100 or less, the repeated Xs may be the same or different, respectively, and if necessary, an alkyleneoxy group represented by the following general formula [2] Ethylene-based unsaturated polymerizable monomer (b)

(Wherein R ₂ represents a hydrogen atom or a methyl group, R ₃ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, Y represents an alkyleneoxy group having 1 to 10 carbon atoms, and q represents 1 to 100) Of the particles in which a layer containing a polymer compound is formed on the surface of the particles by mixing Y, which may be the same or different , and then allowing the polymerization reaction to proceed. On the surface, Ago1, Ago2, Ago3, Ago4, PIWIL1, PIWIL2, PIWIL3, PIWIL4, a carrier on which an antibody against a small RNA binding protein selected from MILI and MIWI is chemically immobilized, and Ago1, Ago2, Ago3, Ago4, A small RNA binding protein selected from PIWIL1, PIWIL2, PIWIL3, PIWIL4, MILI and MIWI and a complex of a small RNA and a small RNA target nucleic acid (small RNA binding protein-small RNA-target nucleic acid complex) The antibody and the small R on the carrier surface Forming a conjugate of NA binding protein-small RNA-target nucleic acid complex;
(2) separating a carrier having a binding product of the obtained antibody and a small RNA binding protein-small RNA-target nucleic acid complex on the surface;
(3) The small RNA-target nucleic acid conjugate is eluted from the conjugate of the antibody and the small RNA binding protein-small RNA-target nucleic acid complex. The method according to claim 15, wherein the antibody is an antibody against Ago1, Ago2, Ago3, or Ago4 .

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